ABSTRACT
The World Health Organization (WHO) has warned that our current arsenal of antibiotics is not innovative enough to face impending infectious diseases, especially those caused by multidrug-resistant Gram-negative pathogens. Although the current preclinical pipeline is well stocked with novel candidates, the last U.S. Food and Drug Administration (FDA)-approved antibiotic with a novel mechanism of action against Gram-negative bacteria was discovered nearly 60 years ago. Of all the antibiotic candidates that initiated investigational new drug (IND) applications in the 2000s, 17% earned FDA approval within 12 years, while an overwhelming 62% were discontinued in that time frame. These "leaks" in the clinical pipeline, where compounds with clinical potential are abandoned during clinical development, indicate that scientific innovations are not reaching the clinic and providing benefits to patients. This is true for not only novel candidates but also candidates from existing antibiotic classes with clinically validated targets. By identifying the sources of the leaks in the clinical pipeline, future developmental efforts can be directed toward strategies that are more likely to flow into clinical use. In this review, we conduct a detailed failure analysis of clinical candidates with Gram-negative activity that have fallen out of the clinical pipeline over the past decade. Although limited by incomplete data disclosure from companies engaging in antibiotic development, we attempt to distill the developmental challenges faced by each discontinued candidate. It is our hope that this insight can help de-risk antibiotic development and bring new, effective antibiotics to the clinic.
Subject(s)
Anti-Bacterial Agents , Gram-Negative Bacterial Infections , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria , Gram-Negative Bacterial Infections/drug therapy , Gram-Negative Bacterial Infections/microbiology , Humans , United States , United States Food and Drug AdministrationABSTRACT
The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.
Subject(s)
Gentamicins , Pharmaceutical Preparations , Aminoglycosides , Animals , Anti-Bacterial Agents , Humans , RatsABSTRACT
Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.
Subject(s)
Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Exodeoxyribonuclease V/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Benzimidazoles/chemistry , Binding Sites , Enzyme Inhibitors/chemistry , Exodeoxyribonuclease V/chemistry , Exodeoxyribonuclease V/genetics , Exodeoxyribonuclease V/metabolism , MutationABSTRACT
New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Multiple, Bacterial/drug effects , Enzyme Inhibitors/pharmacology , Pseudomonas aeruginosa/drug effects , Catalysis/drug effects , Humans , Pseudomonas aeruginosa/metabolismABSTRACT
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30â mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Diynes/pharmacology , Enzyme Inhibitors/pharmacology , Heart/drug effects , Hydroxamic Acids/pharmacology , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/toxicity , Bacterial Proteins/antagonists & inhibitors , Cardiotoxicity , Diynes/chemical synthesis , Diynes/pharmacokinetics , Diynes/toxicity , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/toxicity , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/toxicity , Male , Molecular Structure , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Prodrugs/toxicity , Pseudomonas aeruginosa/drug effects , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Carbon-Nitrogen Ligases/antagonists & inhibitors , Escherichia coli/drug effects , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacterial Outer Membrane/drug effects , Bacterial Outer Membrane/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , Chemical Phenomena , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Microbial Sensitivity Tests , Models, Chemical , Molecular Structure , Mutation , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/geneticsABSTRACT
Conformationally constrained tetracyclic fluoroquinolones (FQs) were synthesized and profiled for their microbiological spectrum. The installation of a seven-membered ring between the pyrrolidine substituents and the C8 position on the FQ core scaffold resulted in a remarkable enhancement of microbiological potency toward both Gram-positive and Gram-negative bacteria. Focused optimization of seven-membered ring composition, stereochemistry, and amine placement led to the discovery of the two lead compounds that were selected for further progression.
Subject(s)
Fluoroquinolones/chemical synthesis , Fluoroquinolones/pharmacology , Tetracyclines/chemical synthesis , Tetracyclines/pharmacology , Acinetobacter baumannii/drug effects , Microbial Sensitivity Tests , Molecular Structure , Pseudomonas aeruginosa/drug effects , Structure-Activity RelationshipABSTRACT
The efficacy of plazomicin for pneumonic plague was evaluated in a non-human primate model. African Green monkeys challenged with a lethal aerosol of Yersinia pestis [median (range) of 98 (15-331) LD50s] received placebo (n=12) or 'humanized' dose regimens (6.25, 12.5 or 25mg/kg every 24h) of plazomicin (n=52) after the onset of fever for a duration of 5 or 10days. All animals treated with placebo died, while 36 plazomicin-treated animals survived through study end. The majority (33/36) were either in the 10-day (high-/mid-/low-dose) or 5-day high-dose groups. The findings suggest an exposure range of plazomicin for treatment of pneumonic/bacteremic Y. pestis infection in humans.
Subject(s)
Disease Models, Animal , Plague/drug therapy , Sisomicin/analogs & derivatives , Animals , Chlorocebus aethiops , Dose-Response Relationship, Drug , Molecular Conformation , Sisomicin/chemistry , Sisomicin/therapeutic useABSTRACT
As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of ß-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.
ABSTRACT
The objective of this study was to evaluate the natural history and pathogenesis of Francisella tularensis in a murine model of inhalational tularemia with the SchuS4 strain. Before the efficacy of antimicrobials could be assessed in this model, further model development was required to determine the optimal time to start therapy. This study helped define the time course of infection after aerosol challenge by quantifying the presence of bacteria in lung, blood, and spleen at multiple harvest points. In this study, mice were infected via a targeted inhaled dose of 100 50% lethal doses (LD50s) (LD50 = 300 CFU) of F. tularensis by whole-body aerosol. At 1, 24, 36, 48, 60, 72, 75, 78, 81, 84, 87, and 90 h postchallenge, groups of 15 animals were sacrificed and blood, lung, and splenic tissue samples were harvested, homogenized, plated, and incubated to evaluate the bacterial load in those tissues. It was determined that of the 3 sample types harvested, splenic tissue provided the most consistent bacterial counts, which steadily increased with the progressing infection. Further, it was determined that lung samples from all (15/15) animals were positive for infection at 75 h postaerosolization and that 14/15 animals had positive splenic tissue counts. Bacterial levels in blood were not predictive of treatment initiation. For future therapeutic evaluation studies in this model using F. tularensis (SchuS4), it was determined that therapy should be initiated at 75 h postchallenge and validated by spleen involvement.
Subject(s)
Bacteremia/microbiology , Francisella tularensis/pathogenicity , Lung/microbiology , Spleen/microbiology , Tularemia/pathology , Aerosols , Animals , Bacterial Load , Disease Models, Animal , Disease Progression , Female , Mice , Mice, Inbred BALB C , Models, TheoreticalABSTRACT
BACKGROUND: Killing of bacterial pathogens by granulocytes is a saturable process, as previously demonstrated. There is virtually no quantitative information about how granulocytes interact with antimicrobial chemotherapy to kill bacterial cells. METHODS: We performed a dose-ranging study with the aminoglycoside plazomicin against Pseudomonas aeruginosa ATCC27853 in a granulocyte-replete murine pneumonia model. Plazomicin was administered in a humanized fashion (ie, administration of decrementing doses 5 times over 24 hours, mimicking a human daily administration profile). Pharmacokinetic profiling was performed in plasma and epithelial lining fluid. All samples were simultaneously analyzed with a population model. Mouse cohorts were treated for 24 hours; other cohorts treated with the same therapy were observed for another 24 hours after therapy cessation, allowing delineation of the therapeutic effect necessary to reduce the bacterial burden to a level below the half-saturation point. RESULTS: The mean bacterial burden (±SD) at which granulocyte-mediated kill was half saturable was 2.45 × 10(6) ± 6.84 × 10(5) colony-forming units of bacteria per gram of tissue (CFU/g). Higher levels of plazomicin exposure reduced the bacterial burden to <5 log10 CFU/g, allowing granulocytes to kill an additional 1.0-1.5 log CFU/g over the subsequent 24 hours. CONCLUSIONS: For patients with large bacterial burdens (eg, individuals with ventilator-requiring hospital-acquired pneumonia), it is imperative to kill ≥2 log10 CFU/g early after treatment initiation, to allow the granulocytes to contribute optimally to bacterial clearance.
Subject(s)
Granulocytes/physiology , Pneumonia, Bacterial/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Sisomicin/analogs & derivatives , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/therapeutic use , Dose-Response Relationship, Drug , Drug Resistance, Bacterial , Female , Mice , Microbial Sensitivity Tests , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/immunology , Pseudomonas Infections/drug therapy , Pseudomonas Infections/immunology , Sisomicin/administration & dosage , Sisomicin/pharmacokinetics , Sisomicin/therapeutic useABSTRACT
After a relatively short untreated interval, pneumonic plague has a mortality approaching 100%. We employed a murine model of aerosol challenge with Yersinia pestis to investigate the early course of pneumonic plague in the lung, blood, and spleen. We fit a mathematical model to all data simultaneously. The model fit to the data was acceptable. The number of organisms in the lung at baseline was estimated to be 135 (median) or 1,184 (mean) CFU/g. The doubling time was estimated as 1.5 to 1.7 h. Between 1 and 12 h postexposure, counts declined, but they then increased by 24 h, a finding hypothesized to be due to innate immunity. The model predicted that innate immunity declined with a half-time of 3 to 3.8 h. The threshold for bacteremia was 6.4 × 10(4) to 1.52 × 10(6) CFU/g. By 42 to 48 h, stationary phase was obtained. Lung bacterial burdens exceeded 10 log CFU/g. Obviating early defenses allows for rapid amplification of Y. pestis in bacteremia, making the rapid course with high mortality understandable.
Subject(s)
Bacteremia/microbiology , Lung/microbiology , Models, Statistical , Plague/microbiology , Spleen/microbiology , Yersinia pestis/immunology , Aerosols , Animals , Bacteremia/immunology , Bacteremia/mortality , Colony Count, Microbial , Female , Immune Evasion , Immunity, Innate , Lung/immunology , Mice , Mice, Inbred BALB C , Plague/immunology , Plague/mortality , Spleen/immunology , Survival Analysis , Yersinia pestis/pathogenicityABSTRACT
The growing threat of untreatable bacterial infections has refocused efforts to identify new antibiotics, especially those acting by novel mechanisms. While the inhibition of pathogen proteases has proven to be a successful strategy for drug development, such inhibitors are often limited by toxicity due to their promiscuous inhibition of homologous and mechanistically related human enzymes. Unlike many protease inhibitors, inhibitors of the essential type I bacterial signal peptidase (SPase) may be more specific and thus less toxic due to the enzyme's unique structure and catalytic mechanism. Recently, the arylomycins and related lipoglycopeptide natural products were isolated and shown to inhibit SPase. The core structure of the arylomycins and lipoglycopeptides consists of a biaryl-linked, N-methylated peptide macrocycle attached to a lipopeptide tail, and in the case of the lipoglycopeptides, a deoxymannose moiety. Herein, we report the first total synthesis of a member of this group of antibiotics, arylomycin A2. The synthesis relies on Suzuki-Miyaura-mediated biaryl coupling, which model studies suggested would be more efficient than a lactamization-based route. Biological studies demonstrate that these compounds are promising antibiotics, especially against Gram-positive pathogens, with activity against S. epidermidis that equals that of the currently prescribed antibiotics. Structural and biological studies suggest that both N-methylation and lipidation may contribute to antibiotic activity, whereas glycosylation appears to be generally less critical. Thus, these studies help identify the determinants of the biological activity of arylomycin A2 and should aid in the design of analogs to further explore and develop this novel class of antibiotic.
Subject(s)
Oligopeptides/chemistry , Oligopeptides/pharmacology , Catalysis , Humans , Hydrogen Bonding , Molecular StructureABSTRACT
Mutation is the driving force behind many processes linked to human disease, including cancer, aging, and the evolution of drug resistance. Mutations have traditionally been considered the inevitable consequence of replicating large genomes with polymerases of finite fidelity. Observations over the past several decades, however, have led to a new perspective on the process of mutagenesis. It has become clear that, under some circumstances, mutagenesis is a regulated process that requires the induction of pro-mutagenic enzymes and that, at least in bacteria, this induction may facilitate evolution. Herein, we review what is known about induced mutagenesis in bacteria as well as evidence that it contributes to the evolution of antibiotic resistance. Finally, we discuss the possibility that components of induced mutation pathways might be targeted for inhibition as a novel therapeutic strategy to prevent the evolution of antibiotic resistance.
Subject(s)
Drug Resistance, Bacterial/genetics , Evolution, Molecular , Mutation , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Humans , Models, Biological , MutagenesisABSTRACT
Staphylococcus aureus infections can be difficult to treat due to both multidrug resistance and the organism's remarkable ability to persist in the host. Persistence and the evolution of resistance may be related to several complex regulatory networks, such as the SOS response, which modifies transcription in response to environmental stress. To understand how S. aureus persists during antibiotic therapy and eventually emerges resistant, we characterized its global transcriptional response to ciprofloxacin. We found that ciprofloxacin induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle. In addition, we found that ciprofloxacin induces the SOS response, which we show, by comparison of a wild-type strain and a non-SOS-inducible lexA mutant strain, includes the derepression of 16 genes. While the SOS response of S. aureus is much more limited than those of Escherichia coli and Bacillus subtilis, it is similar to that of Pseudomonas aeruginosa and includes RecA, LexA, several hypothetical proteins, and a likely error-prone Y family polymerase whose homologs in other bacteria are required for induced mutation. We also examined induced mutation and found that either the inability to derepress the SOS response or the lack of the LexA-regulated polymerase renders S. aureus unable to evolve antibiotic resistance in vitro in response to UV damage. The data suggest that up-regulation of the tricarboxylic acid cycle and induced mutation facilitate S. aureus persistence and evolution of resistance during antibiotic therapy.
Subject(s)
Ciprofloxacin/pharmacology , SOS Response, Genetics/physiology , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Citric Acid Cycle/genetics , Citric Acid Cycle/physiology , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial , Mutation , Oligonucleotide Array Sequence Analysis/methods , Reverse Transcriptase Polymerase Chain Reaction , SOS Response, Genetics/genetics , Staphylococcus aureus/metabolism , Staphylococcus aureus/radiation effects , Ultraviolet RaysABSTRACT
Pseudomonas aeruginosa infections can be virtually impossible to eradicate, and the evolution of resistance during antibiotic therapy is a significant concern. In this study, we use DNA microarrays to characterize the global transcriptional response of P. aeruginosa to clinical-like doses of the antibiotic ciprofloxacin and also to determine the component that is regulated by LexA cleavage and the SOS response. We find that genes involved in virtually every facet of metabolism are down-regulated in response to ciprofloxacin. The LexA-controlled SOS regulon identified by microarray analysis includes only 15 genes but does include several genes that encode proteins involved in recombination and replication, including two inducible polymerases known to play a role in mutation and the evolution of antibiotic resistance in other organisms. The data suggest that the inhibition of LexA cleavage during therapy might help combat this pathogen by decreasing its ability to adapt and evolve resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Ciprofloxacin/pharmacology , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/genetics , SOS Response, Genetics/genetics , Bacterial Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Gene Expression Profiling , Genes, Bacterial , Oligonucleotide Array Sequence Analysis , Pseudomonas aeruginosa/drug effects , RNA, Bacterial/analysis , RNA, Bacterial/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Recombinases/genetics , Regulon , Serine Endopeptidases/geneticsSubject(s)
Anti-Bacterial Agents/pharmacology , DNA/pharmacokinetics , Evolution, Molecular , Genes, Bacterial , Streptococcus pneumoniae/drug effects , Chromosomes, Bacterial/genetics , Drug Resistance, Microbial , Models, Genetic , Recombination, Genetic , Streptococcus pneumoniae/genetics , Transformation, GeneticABSTRACT
The emergence of drug-resistant bacteria poses a serious threat to human health. Bacteria often acquire resistance from a mutation of chromosomal genes during therapy. We have recently shown that the evolution of resistance to ciprofloxacin in vivo and in vitro requires the induction of a mutation that is mediated by the cleavage of the SOS repressor LexA and the associated derepression of three specialized DNA polymerases (polymerase II [Pol II], Pol IV, and Pol V). These results led us to suggest that it may be possible to design drugs to inhibit these proteins and that such drugs might be coadministered with antibiotics to prevent mutation and the evolution of resistance. For the approach to be feasible, there must not be any mechanisms through which bacteria can induce mutations and acquire antibiotic resistance that are independent of LexA and its repressed polymerases. Perhaps the most commonly cited mechanism to elevate bacterial mutation rates is the inactivation of methyl-directed mismatch repair (MMR). However, it is unclear whether this represents a LexA-independent mechanism or if the mutations that arise in MMR-deficient hypermutator strains are also dependent on LexA cleavage and polymerase derepression. In this work, we show that LexA cleavage and polymerase derepression are required for the evolution of clinically significant resistance in MMR-defective Escherichia coli. Thus, drugs that inhibit the proteins responsible for induced mutations are expected to efficiently prevent the evolution of resistance, even in MMR-deficient hypermutator strains.
Subject(s)
Ciprofloxacin/pharmacology , DNA Replication/drug effects , Drug Resistance, Bacterial/physiology , Escherichia coli Proteins/physiology , Escherichia coli/drug effects , SOS Response, Genetics/physiology , Bacterial Proteins , Base Pair Mismatch/genetics , DNA Repair/drug effects , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , MutS DNA Mismatch-Binding Protein/genetics , Mutation , Serine EndopeptidasesABSTRACT
The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.